| Summary: | This research aims to determine the characteristics of extra-cellular protease enzymes derived from the intestines of sea cucumbers as a candidate for bio-detergent, to optimize the growth of extra-cellular protease-producing bacteria and to identify the enzymes producing bacteria by DNA sequencing. The study consisted of 5 stages, namely isolation, selection and identification of extra-cellular protease-producing bacterial enzymes, bacterial growth optimization test in the production of extra-cellular protease enzymes, enzyme isolation and characterization, enzyme ability testing of various detergent components and determining the highest conditions of enzyme activity, and purification and identification from the extra-cellular protease enzyme. The results showed that bacteria isolated from the sea cucumbers <em>Stichopus hermanni</em>, <em>Holothuria atra</em> and <em>H.leucopilota.</em> Bacterial isolation derived from the contents of all these sea cucumbers produced 60 isolates and 22 isolates were active against the enzyme protease. Optimization test of carbon compounds (C), namely glucose, fructose, and molasses produces the enzyme molasses. Nitrate (N) compounds optimization test, namely: Ammonium chloride, Ammonium nitrate, Urea found Ammonium nitrate as the best. Optimization test results of the concentration of substances C, N and optimization on the parameters of salinity (25, 30, 35 ppt), pH (6, 7, 8) and temperature (25, 30, 35℃), produced the best salinity at 30 ppt, the best ph at 8 and the best temperature at 30 ℃. The results of the fermentation test showed that protein hydrolysis in TH.IP.4 isolate media was better than that in THDM.IP.3. There was a positive correlation between cell density and the presence of protease enzymes, where the higher the number of cells, the higher the production of protease enzymes. Protease activity in TH.IP.4 isolates was better than THDM.IP.3 isolates. Molecular identification results showed that isolate 0TH.IP.4 had the closest match (99%) with <em>Bacillus cereus</em> and THDM.IP.3 was a complete match (100%) with <em>Bacillus thuringiensis</em>.
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